Figure 6.

Administration of Casp3Inh to mice and cultured human BMSSCs. (A) BMD in Casp3Inh-treated mice. X-ray images show bone density differences in femora between Casp3Inh-treated mice and DMSO-treated mice after both OVX and sham operation (left). Arrows and asterisks represent cortical and trabecular bone area in femora, respectively. pQCT analysis also showed decreased cancellous BMD of distal femora in Casp3Inh-treated mice, especially after OVX (right). Error bars represent the mean ± SD (n = 4; P < 0.01; ^#P <0.05). (B) Western blot analysis of human BMSSCs. Casp3Inh-treated BMSSCs showed upregulated expression of TGF-βRI and TGF-βRII compared with DMSO-treated BMSSCs, especially after OVX. After TGF-β treatment, the expression of Smad2 and p-Smad2 was increased both in Casp3Inh-OVX and DMSO-OVX BMSSCs. Even without TGF-β treatment, p-Smad2 was detectable in BMSSCs from Casp3Inh-OVX mice. (C) Caspase-3 activity in human BMSSCs. After BMSSCs were treated with staurosporin (STS), caspase-3 activity was detected (upper panel, green staining, arrows). Caspase-3 activity was not detectable with pretreatment with Casp3Inh (lower panel). Blue color represents nuclei staining with DAPI. Original magnification, ×200. (D) Alizarin red staining of human BMSSCs. BMSSCs were treated with Casp3Inh or DMSO for 24 hours and then cultured under the osteogenic inductive condition. Calcium accumulation was decreased in Casp3Inh-treated BMSSCs (left). Matrix calcium levels released by acid treatment were measured (right). Error bars represent the mean ± SD (n = 4; P < 0.01). (E) Bone formation by human BMSSCs in vivo. Bone formation assessed by H&E staining was decreased in the Casp3Inh-treated BMSSC transplant (left). Original magnification, ×200. The BFR was calculated as the percentage of newly formed bone area per total area of transplant at the representative cross-sections. Error bars represent the mean ± SD (right panel; n = 5; P < 0.01).